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| [[Image:MARIMOD3ca83c.png| 150px]]
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| [[Biomod/2013/Hokkaido | <font face="trebuchet ms" style="color:#FFFFFF" size="4"> '''Home''' </font>]]
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| [["What's "Marimo"?" | <font face="trebuchet ms" style="color:#FFFFFF" size="4"> '''What's Marimo''' </font>]]
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| [["Design" | <font face="trebuchet ms" style="color:#FFFFFF" size="4">'''Design''' </font>]]
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| [["Results" | <font face="trebuchet ms" style="color:#FFFFFF" size="4"> '''Results''' </font>]]
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| <br>
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| [["Materials & Methods" | <font face="trebuchet ms" style="color:#FFFFFF" size="4"> '''Materials & Methods''' </font>]]
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| [["People" | <font face="trebuchet ms" style="color:#FFFFFF" size="4">'''People''' </font>]]
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| [["Sponsors" | <font face="trebuchet ms" style="color:#FFFFFF" size="4">'''Sponsors''' </font>]]
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| </div>
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| ----
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| ===[[Image:大見出し.png| 30px]] <font size="5">Preparation of Ring-Shaped Microtubule Assemblies</font>===
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| ====[[Image:小見出し.png|18px]] <font size="4">Purification of tubulin</font>====
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| <p>
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| Tubulin was purified from porcine brain through two cycles of polymerization-depolymerization <br>
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| using a high-molarity buffer (1 M PIPES, 20 mM EGTA, 10 mM MgCl<sub>2</sub>; pH adjusted to 6.8); <br>
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| high-molarity PIPES buffer (HMPB).
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| </p>
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| <p>
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| <font size="3">1.Preparation of Brain homogenate</font>
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| ----
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| Porcine brains were purchased from a local slaughterhouse, and conserved before<br>
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| use in ice-cold PBS (20 mM Na-phosphate, 150 mM NaCl, pH7.2). Brains were<br>
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| cleaned of blood clots with , weighed, and transferred to a homogenizer (nissei, AM-7).<br>
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| Cold (4 °C) depolymerization buffer (DB) (50 mM 2-[N-morpholino] ethanesulfonic acid,<br>
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| 1 mM CaCl<sub>2</sub>, pH 6.6) was added at a ratio of 1 mL/g of brain tissue. The mixture<br>
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| was homogenized eight times at 50000 rpm for 15 s.<br>
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| </p>
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|
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| <p>
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| <font size="3">2.First cold and warm spin</font>
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| ----
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| Brain homogenates were then centrifuged at 12,000 rpm for 60 min at 4 °C. <br>
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| The supernatants were pooled and supplemented with an equal volume of warm (37 °C) <br>
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| high-molarity PIPES buffer (HMPB) (1 M PIPES, 10 mM MgCl<sub>2</sub>, and 20 mM EGTA, pH 6.9) ,<br>
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| ATP (1.5 mM final), and GTP (0.5 mM final). ATP is included in this step to <br>
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| remove motors and other proteins, which bind to microtubules in an ATP sensitive manner [2].<br>
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| An equal volume (1/3 of the final volume) of pre-warmed to 37 °C anhydrous glycerol <br>
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| was added to this solution. This mixture was incubated in a 37 °C water bath for 1h. <br>
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| The polymerized tubulin was then centrifuged at 44,000 rpm for 30 min at 37 °C.
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| </p>
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| <p>
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| <font size="3">3.Second cold and warm spin</font>
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| ----
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| The resulting microtubule pellets were resuspended in 100 mL of cold DB. <br>
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| The depolymerized tubulin was subsequently centrifuged in at 34,000 rpm for 30 min at 4 °C.<br>
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| The supernatant from this centrifugation step was mixed with an equal volume <br>
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| of the HMPB supplemented with ATP and GTP, followed by the addition of glycerol<br>
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| (1/3 of final volume) as described above. The mixture was incubated in a 37 °C<br>
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| water bath for 1h, and the polymerized microtubules centrifuged in at 44,000 rpm <br>
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| for 30 min at 37 °C. Following the centrifugation, the microtubule pellets were resuspended <br>
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| in 15 mL of ice-cold BRB80 (80 mM PIPES, 1 mM MgCl<sub>2</sub>, 1 mM EGTA, pH 6.8) and then <br>
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| incubated for a further 10 min on ice. After this polymerization step, the tubulin <br>
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| was centrifuged in at 35,000 rpm for 30 min at 4 °C. The supernatant was collected <br>
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| and snap-frozen in 100 µL aliquots in liquid nitrogen.
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| </p>
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| <p>
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| <font size="3">4.Determination of tubulin concentration</font>
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| ----
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| Tubulin concentration was determined by SDS-PAGE.
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| </p>
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| ====[[Image:小見出し.png|18px]] <font size="4">Preparation of biotin labeled tubulin</font>====
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| <p>
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| 1. Tubulin was thawed, and polymerized at 37 °C for 15 min.<br>
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| 2. Biotin-XX-SE (Molecular Probes, Cat. B-1606) was dissolved at 0.1 M in dry dimethyl sulfoxide (DMSO). <br>
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| 3. Biotin-XX-SE solution was added to tubulin, while pipetting to distribute it rapidly to a final concentration of 2 mM. incubate at 37 °C for 20 min.<br>
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| 4. Mixture was layered onto cushions and spun at 54,000 rpm for 1h at 37 °C.<br>
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| 5. Pellets were resuspended and spun cold, being careful to wash the cushion inter face well to remove all the biotin-XX-SE.<br>
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| 6. Tubulin was depolymerized and spun 40,000 rpm for 15 min at 4 °C.<br>
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| 7. Steps (3) – (6) were performed to give once cycled biotin-tubulin.<br>
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| 8. Steps (3) – (6) were repeated to give twice cycled biotin-tubulin. The final pellet is resuspended in BRB80.<br>
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| 9. The final biotin-tubulin is frozen and stored as per the cycled tubulin.<br>
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| </p>
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| <p>
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| =====<font size="3">Determination of stoichiometry</font>=====
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| Tubulin concentration was determined by SDS-PAGE electrophores.<br>
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| To quantify biotin, we use defference of avidin and avidin-biotin complex in <br>
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| spectroscopic characteristic because avidin combines stoichiometrically with biotin.<br>
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| The dye 4-hydroxyazobenzene-2’-carboxylic acid (HABA), which binds only to avidin with changing spectrum,<br>
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| so that it can be used as an indicator for unoccupied binding sites [1].<br>
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| </p>
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|
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| <p>
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| <font size="3">1.Prepare standard curve.</font><br>
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| ----
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| Biotin standard solutions [(0, 10, 20, 50, 100, 200, 300, 500 µM) biotin,<br>
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| 80 mM PIPES, 5 mM MgCl<sub>2</sub>, 1 mM EGTA] were prepared.<br>
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| Each biotin standard solutions was added to avidin solution of which final concentration is<br>
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| 0.4 mg mL<sup>-1</sup> avidin, 250 mM HABA, 80 mM PIPES, 5 mM MgCl<sub>2</sub>, 1 mM EGTA.<br>
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| The mixtures were left for 15 min at roomtemperature.<br>
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| A<sub>500</sub> of these solutions were measured to prepare standard curve.
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| <br><br>
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|
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| <font size="3">2.Determination of biotin.</font><br>
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| ----
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| 1 mg mL<sup>-1</sup> Pronase was added to biotin-labeled tubulin, and left for 1 h at 37 °C.<br>
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| This mixture was added to avidin solution, and left for 15 min at roomtemperature.<br>
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| A<sub>500</sub> of this solution were measured to determine concentration of biotin.<br>
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|
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| ====<font size="4">Preparation of Microtubule</font>====
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| Biotinylated- and rhodamine-labeled MTs were obtained by polymerizing biotin–tubulin <br>
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| and rhodamine–tubulin (60% byotinilated and 10% rhodamine-tubulin; final tubulin concentration, 42 mM);<br>
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| the solution containing the MTs was then diluted with motility buffer (80 mM PIPES, 1 mMEGTA,<br>
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| 2 mM MgCl<sub>2</sub>, 0.5 mg mL<sup>-1</sup> casein, 1 mM DTT, 4.5 mg mL<sup>-1</sup> D-glucose,<br>
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| 50 U mL<sup>-1</sup> glucose oxidase, 50 U mL<sup>-1</sup> catalase, 10 mM paclitaxel,<br>
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| and 1% DMSO; pH 6.8).
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|
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|
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| ====<font size="4">Dynamic self-assembly</font>====
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| Flow-cells were prepared by placing a coverglass on a slideglass (26 × 76 mm2) equipped with<br>
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| a pair of double-sided tape to form a chamber of approximately 4 × 18 × 0.1 mm<sup>3</sup> (W × L × H) in dimension.<br>
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| The flow cell was filled with 0.2 mg mL<sup>-1</sup> anti-GFP antibody (Invitrogen) for 3 min,<br>
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| followed by a wash with 5 µL of casein solution (80 mM PIPES, 1 mM EGTA, 1 mM MgCl<sub>2</sub>, 0.5 mg mL<sup>-1</sup> casein; pH adjusted to 6.8 using HCl). <br>
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| After incubating for 3 min with casein solution to mask the remaining glass surface, 5 µL of 200 nM kinesin solution <br>
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| (80 mM PIPES, 40 mM NaCl, 1 mM EGTA, 1 mM MgCl<sub>2</sub>, 0.5 mg mL<sup>-1</sup> casein, 1 mM DTT, 4.5 mg mL<sup>-1</sup> D-glucose,<br>
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| 50 U mL<sup>-1</sup> glucose oxidase, 50 U mL<sup>-1</sup> catalase, 10 mM paclitaxel, 1% DMSO; pH 6.8) <br>
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| were introduced and incubated for 3 min to bind the kinesins to the antibody. The flow cell<br>
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| was washed with 3 mL of motility buffer. 3 mL of MTs solution (5000 nM in motility buffer, Bt 3000 nM) was then <br>
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| introduced and incubated for 3 min, followed by washing with 5 µL of motility buffer.<br>
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| A diluted solution (5 µL) of streptavidin (300 nM in motility buffer) was then introduced <br>
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| and incubated for 3 min, followed by washing with 5 µL of motility buffer. Finally, Dynamic<br>
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| self-assembly was initiated by applying 5 µL of ATP solution (motility buffer supplemented with 5 mM ATP).
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| This experiment was performed at room temperature.<br><br><br>
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| [[Image:リングの形成.png | 400px]]
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|
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| <br><br><br><br>
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| ----
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| ===[[Image:大見出し.png| 30px]] <font size="5">Preparation of Marimo-Gel</font>===
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| ====[[Image:小見出し.png| 18px]] <font size="4">Preparation of spinach thylakoid membranes</font>====
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| <p>
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| All purification procedures were performed at 4 °C. <br>
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| Thylakoid membranes were prepared from spinach leaves by the modified method of Yu et al.<br>
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| The spinach leaves ( ～160 g) were washed with deionized water and homogenized in 500 mL of homogenization solution<br>
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| (0.3 M sucrose, 20 mM NaCl, 5 mM MgCl<sub>2</sub>, 50 mM Tris-HCl, pH was adjusted to 7.6)<br>
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| for 40 sec using an AM-10 homogenizer (Nihon Seiki Seisakusho, Japan). <br>
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| The homogenate was filtered through four-time folded gauze.<br>
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| The flow through was suspended in 400 mL of a high ionic strength buffer (10 mM Hepes-KOH, 150 mM NaCl, pH8).<br>
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| The suspension was centrifuged at 10,000g for 20 min and the precipitate was resuspended in 15 mL<br>
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| of homogenization solution including 5% DMSO and flash frozen in liquid nitrogen,<br>
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| and stored in liquid nitrogen.
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| </p>
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| <p>(Yu A. H. C; Hosono K. ''Biotechnol. Lett.'' '''1991''', ''13'', 411.)</p>
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| ====[[Image:小見出し.png| 18px]] <font size="4">Entrapment of thylakoid membranes in alginate beads</font>====
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| <p>
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| This procedure is based on the method described by Paul F. et al. and Zekorn T. et al.<br>
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| A 0.3 mL of suspension of thylakoid membrane containing 1.41 mg protein/mL and 2.7 mL of<br>
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| a 2%(w/v) sodium alginate solution (100 mM K<sub>3</sub>PO<sub>4</sub>, 100 mM MgCl<sub>2</sub>, 100 mM NaCl, 500 mM PIPES,<br>
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| 200 mM ADP, pH was adjusted to 7.6) were placed in a 1 mL syringe. Using syringe pump (HA2000P, Harvard apparatus, US),<br>
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| the alginate solution including thylakoid membrane was slowly ejected from the needle and was blown by a nitrogen gas.<br>
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| The tear shaped green droplet firstly encountered mineral oil phase and transformed into globular shape. <br>
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| Then the droplet sunk into the second phase, which contains 50 mM BaCl<sub>2</sub> and cross-linkage of <br>
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| alginate with barium occurred. Because leak was not observed even after few weeks from the encapsulation,<br>
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| the cross-linked alginate mesh seemed to be enough small to support thylakoid membranes.<br>
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| </p>
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| <p>
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| (Paul F.; Vignais P. M. ''Enzyme Mcrob. Technol.'' '''1980''', ''2'', 281.)<br>
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| (Zekron T.; Horcher A.; Siebers U.; Schnettler R.; Klock G.; Hering B.; Zimmermann U.;<br>
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| Bretzel R. G.; Federlin K. ''Acta Diabetol'', '''1992''', ''29'', 99.)
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| </p>
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| <br><br><br>
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| ----
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|
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| ===[[Image:大見出し.png| 30px]] <font size="5">Preparation of Micrometer-sized Gear</font>===
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| <p>
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| First, Lift-off Layer (LOL) layer and SU-8 layer were placed on a silicon substrate. <br>
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| Micro gears were drawn on SU-8 layer by using laser etching technique. Micro gears were<br>
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| separated from silicon substrate by using alkaline developer (NMD-3) which can dissolve LOL layer. <br>
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| After removal of micro gears from silicon substrate were collected by centrifugation.<br>
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| </p>
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| [[Image:Gear.png| 500px]]
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|
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| <br><br><br>
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|
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| ===[[Image:大見出し.png| 30px]] <font size="5">Measurement the power of rotational motion of ring</font>===
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| <p>
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| To measure the force of rotational motion of MT ring, we used optical tweezers. After MT ring preparation, we washed <br>
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| the flow cell with motility buffer and put the polystyrene bead by biotin-streptavidin specific interaction. <br>
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| Polystyrene bead was trapped with laser beam and measured the rotating power of MT ring.
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| </p>
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| [[Image:Measurement_force_of_ring.jpg| 400px]]
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|
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| ----
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| ===[[Image:大見出し.png| 30px]] <font size="5">Devices</font>===
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| ====[[Image:小見出し.png| 18px]] <font size="4">Microscope</font>====
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| <p>
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| To study the motility of MTs, we used a 100 W mercury lamp for illuminating of samples and<br>
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| an epifluorescence microscope (Eclipse Ti, Nikon) using an oil-coupled Plan Apo 60×objective (Nikon)<br>
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| for visualizing of samples. Also we used UV cut-off filter blocks (GFP-HQ: EX455-485, DM495, BA500-545; Nikon)<br>
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| in the optical path of the microscope; these blocks allowed visualization of samples but eliminated the UV portion of the radiation, thus minimizing the harmful effect of UV radiation on the samples.<br>
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| Moreover we connected a cooled-CMOS camera (NEO sCMOS, Andor) to a PC for capturing images.
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| </p>
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| <p>
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| [[Image:Microscope.gif| 400px]]
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| </p>
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|
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| ====[[Image:小見出し.png| 18px]] <font size="4">Optical tweezer</font>====
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| <p>Optical tweezer is a scientific instruments which can hold and move microscopic objects <br>
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| by using a highly focused laser beam. It provides an attractive or repulsive force (typically on the order of pN),<br>
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| depending on the refractive index mismatch. In this work, we will measure the force of<br>
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| micro gear by using an optical tweezer.<br>
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| Here we use a Nd:YAG laser (1064 nm wavelength) to trap a biological specimens. <br>
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| This is because such specimens (being mostly water) have a low absorption coefficient at this wavelength. <br>
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| A low absorption is desirable so as to minimize damage of the biological specimens, <br>
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| which sometimes referred to as opticution. Moreover He:Ne Pilot laser(633 nm wavelength) <br>
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| was used with Nd:YAG laser for visualization of specimens. <br>
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| Capturing images were sent to a PC and edited by imaging software (Nikon NIS Elements).</p>
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| [[Image:Optical tweezer.jpg| 400px]]
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| [[Image:Optical_tweezer_device.png| 400px]]
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